Project Summary This R35 application describes a robust research framework to facilitate discovery and translation of new therapeutic targets in Acute Respiratory Distress Syndrome (ARDS), a common cause of acute respiratory failure that carries a high mortality rate and has no beneficial targeted therapies. ARDS remains a significant health problem affecting 190,000 Americans per year, costing billions of dollars, and leaving the majority of patients dead or significantly impaired. New insights into the pathogenesis are needed to deepen our understanding of the underlying mechanisms that lead to ARDS as well as to develop novel therapeutics. Thus, there is an unmet need to define clinically relevant therapeutic targets that can be studied mechanistically and rapidly carried through robust pre-clinical studies. For the last 14 years I have been building my research team to be on the forefront of translational discovery. My group has made major contributions to understanding ARDS pathophysiology. The major focus of my laboratory is defining the key cellular and molecular regulators of alveolar capillary barrier function and dysfunction that underlies ARDS pathology. My R35 research program is designed to identify novel mediators in ARDS using lung tissue imaging mass spectrometry and deep phenotyping of gene expression profiles at the single cell level coupled with advanced statistical methods to identify leading targets. New targets will be studied in in vitro transgenic model systems to define cellular and molecular mechanisms in order to facilitate the development of novel therapeutics to be tested in pre-clinical models. My research framework is centered on three goals: Discovery, Mechanism and Translation. Goal 1 ? Discovery. To accomplish this goal, we will break new ground using imaging mass spectrometry and single cell RNA sequencing to create an expression profile and protein ?map? of the injured and uninjured human lung. Using advanced statistical approaches, we will identify promising targets to take forward into further studies. Goal 2 ? Mechanism. Leveraging our existing lung injury models, institutional resources and new approaches, we will define the fundamental pathologic mechanisms that lead to alveolar capillary barrier permeability in ARDS by generating novel transgenic cell and mouse lines for mechanistic studies. Goal 3 ? Translation. Building on our existing ex vivo human lung and in vivo mouse models, we will generate rigorous pre-clinical data based on new targets identified in our Discovery and Mechanism studies. With this R35, my lab will advance the mission of the NHLBI by: generating a catalogue of single cell transcription profiles and tissue protein expression levels in acutely injured and uninjured human lung, identifying novel therapeutic targets in ARDS, defining the cellular and molecular mechanisms regulating alveolar capillary barrier dysfunction and conducting pre-clinical studies in mouse and human models. The R35 will provide the support and flexibility necessary for me to break new ground in ARDS.